A natural polymorphism in b-lactamase is a global suppressor (antibiotic resistanceyprotein evolution)

A M182T substitution was discovered as a second-site suppressor of a missense mutation in TEM-1 b-lactamase. The combination of the M182T substitution with other substitutions in the enzyme indicates the M182T substitution is a global suppressor of missense mutations in b-lactamase. The M182T substitution also is found in natural variants of TEM-1 b-lactamase with altered substrate specificity that have evolved in response to antibiotic therapy. The M182T substitution may have been selected in natural isolates as a suppressor of folding or stability defects resulting from mutations associated with drug resistance. This pathway of protein evolution may occur in other targets of antimicrobial drugs such as the HIV protease. The production of b-lactamase is the principal mechanism of bacterial resistance to b-lactam antibiotics, such as penicillins and cephalosporins. b-lactamase provides resistance by catalyzing the hydrolysis of b-lactam antibiotics to ineffective antimicrobials. TEM-1 b-lactamase is the most prevalent plasmid-mediated b-lactamase in Gram-negative bacteria (1). It is able to efficiently hydrolyze penicillins and most cephalosporins, but not the more recently developed extendedspectrum cephalosporins, such as ceftazidime and cefotaxime (2). In addition, b-lactamase inhibitor compounds, such as clavulanic acid, have been developed. These compounds themselves do not possess antimicrobial activity but instead are used in conjunction with other b-lactams, such as ampicillin (3). Soon after the introduction of extended-spectrum cephalosporins and inhibitors there were reports of transferable resistance to the drugs (4). Cloning and DNA sequencing revealed that much of the resistance was due to TEM blactamase enzymes that contained 1–4 amino acid substitutions (4). These substitutions alter the substrate profile of the enzyme such that it can hydrolyze the extended-spectrum cephalosporins or is insensitive to b-lactamase inhibitors (4, 5). Interestingly, most of the substitutions found in inhibitorresistant enzymes are different from those found in enzymes able to cleave extended-spectrum cephalosporins (5). An exception appears to be the M182T substitution, which has been identified in both inhibitor-resistant enzymes (TEM-32) and extended-spectrum b-lactamases (TEM-43) (refs. 6 and 7; K. Bush, personal communication). In this study, the M182T substitution was identified as a second-site suppressor of an asparagine for leucine substitution at position 76, which is buried in the TEM-1 structure. Further experiments demonstrated the M182T substitution can suppress the effects of deleterious substitutions at other sites in the protein. These findings suggest that the M182T substitution acts as a global suppressor of b-lactamase substitutions that disrupt the folding andyor stability of the enzyme. MATERIALS AND METHODS Strains and Plasmids. Escherichia coli BW313 [Hfr lysA(61– 62) dut1 ung1 thi1 recA1 spoT1] was used to propagate plasmid DNA before oligonucleotide-directed mutagenesis (8). Mutagenized DNA was initially introduced into E. coli ES1301 [lacZ53 mutS201::Tn5 thyA36 rha5 metB1 deoCIN(rrnD-rrnE)] (9). E. coli ES1301 also was used as the mutator strain in reversion analysis. E. coli XL1-Blue [recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F9::Tn10 (Tetr) proAB DlacIq (lacZ)M15]] was used as the host for the assay of antibiotic susceptibility, for immunoblotting, for specific activity measurements, and for the preparation of single-stranded DNA (10). E. coli SB646 [DfhuA Dptr DdegP DompT Dprc::kan] is a protease-deficient strain that was used in immunoblotting and specific activity experiments. This strain was a gift of Steve Bass (Genentech). Mutagenesis was performed on the plasmid pBG66, which was used in previous studies (11). The pBG66 plasmid contains the wild-type blaTEM-1 gene and a cat gene encoding chloramphenicol acetyltransferase. This 4.8-kb plasmid also contains the ColEI and f1 origins of DNA replication. Construction of Mutants. The L76N and L76S substitutions were constructed by oligonucleotide-directed mutagenesis using the method of Kunkel et al. (8). The L76 codon was randomized using the following oligonucleotide, where S represents C or G and N represents a mixture of all four nucleotides: L76X 59-AATACCGCGCCACASNNCAGAACTTTAAAAGTG-39. The template for mutagenesis was the pBG66 plasmid containing a SalI linker insertion into codon 76 of the blaTEM-1 gene (12). In addition, a deletion of two nucleotides from codon 76 created a frameshift mutation and rendered this mutant nonfunctional. The L76X oligonucleotide was annealed to a single-stranded DNA template of the SalI insertion mutant, and mutagenesis was performed exactly as described by Huang et al. (12). The L76N and L76S substitutions were identified by DNA sequencing a collection of 40 mutants. The M182T single mutant was constructed by digesting the pBG66 plasmid containing the L76N:M182T double substitution with HincII and EcoRI which releases a DNA fragment containing the L76 codon, but not the M182 codon. A HincIIEcoRI fragment containing the wild-type L76 codon then was used to replace the released fragment. The I47Y:E48C mutant was selected from the L46–48 random library of mutants (12). The mutant was selected by transforming the L46–48 plasmid library into E. coli XL1-Blue and spreading the transformed cells on Luria–Bertani (LB) agar supplemented with 12.5 mgyml chloramphenicol. Individual colonies then were picked and patched onto agar plates containing either 1 mgyml or 100 mgyml ampicillin. The I47Y:E48C mutant was picked for DNA sequencing, and further characterization was based on the fact that it grew on plates with 100 mgyml, but not 1 mgyml, ampicillin. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y948801-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: LB, Luria–Bertani; MIC, minimal inhibitory concentration. ‡To whom reprint requests should be addressed.